Effect Of LPS-TLR4 Complex Internalization On LPS-activated Macrophages And Its Mechanism Study

Sepsis is systemic inflammatory response syndrome (SIRS) with an infectious origin. Sepsis may progress into septic shock and multiple organ dysfunction syndrome (MODS) which ultimately threaten the pathients'lives. Lipopolysaccharides (LPS) are one component of the cell walls of Gram-negative bacteria. LPS are regarded as the major pathogen associated molecule pattern (PAMP) which activate the inflammatory signal transduction pathway, upregulate the expression of inflammatory cytokines (TNF-α, IL-6, etc.) in innate immune cells and cause sepsis and septic shock. Toll-like receptor 4, a kind of typeⅠtrans-membrane receptors, acts as the pattern recognition receptors (PRR) for LPS and plays important roles in initiating the recognition and consequent reactions of LPS in mononuclear phagocyte system. It is conventionally thought that the recognition of TLR4 with LPS occurres on the surface of monocytes/macrophages. LPS-induced TLR4 internalization mainly contributes to the degradation of the LPS-TLR4 complex as well as the desensitization of the activated cells. However, recent evidences indicated that internalization of LPS and TLR4 may be also relevant to the activation of the monocytes/macrophages. However, little was known about the mechanisms and functional consequences of the endocytic uptake of the LPS-TLR4 complex. So we took this study to investigate the internalizing pathways of LPS-TLR4 complex and its effect on cell activation triggered by LPS in macrophages.ObjectTo establish the cell model of internalization dysfuction and examine the effect on internalization of LPS-TLR4 complex in this cell model and the related mechanisms.Methods1. Establishment of RAW264.7 cells model of internalization dysfuction(1) Utilizing chemical inhibitors, we established cell model of internalization dysfunction in a murine macrophage cell line RAW264.7cells. The inhibition effectiveness on LPS-TLR4 complex internalization was examined by Flow Cytometry and confocal laser scaning microscopy①Clathrin inhibitor MDC MDC specifically inhibited the function of clathrin and was utilized to establish an internalization dysfunction model in RAW264.7 cells.②Dynamin inhibitor dynasore Dynasore specifically inhibited the function of dynamin and was also utilized to establish an internalization dysfunction model in RAW264.7 cells.③Endosome acidifying maturation inhibitor chlorquine(CQ) Chlorquine inhibited endosome maturation and was utilized to establish endosome internalization dysfunction in RAW264.7 cells.(2) clathrin siRNA siRNA technology was utilized for the knockdown of clathrin to establish an internalization dysfunction model in RAW264.7 cells.2. Examination of the activation status of RAW264.7 cells model of internalization dysfunctionRAW264.7 cells were incubated in the presence or absence of MDC(100μM) or dynasore(80μM) or CQ(20μg/ml) for 1 hours, and then stimulated with LPS (100ng/ml) for 2h or 24h. After 2h, the total RNA were extracted and the RT-PCR assay was applied to check the expression of TNF-αand IL-6 mRNA in normal cells and model cells; After 24h, and cytokine secretion in the cell-free culture supernatants was evaluated using ELISA or Cytokine antibody array ;3. The mechanism study of internalization dysfunction on the activation of RAW264.7 cells(1) RAW264.7 cells were incubated in the presence or absence of MDC(100μM) or dynasore(80μM) or CQ(20μg/ml) for 1 hours, and then stimulated with LPS (100ng/ml) for 2h . MyD88 mRNA and TRAM mRNA expressions were detected using RT-PCR.(2) RAW264.7 cells were incubated in the presence or absence of MDC(100μM) or dynasore(80μM) or CQ(20μg/ml) for 1 hours, and then stimulated with LPS (100ng/ml) for 4h and 12h, total nucleic protein were extracted and NF-κB activation condition was detected using ELISA;(3) RAW264.7 cells were incubated in the presence or absence of MDC(100μM) or dynasore(80μM) or CQ(20μg/ml) and then stimulated with LPS (100ng/ml) for 12h, levels of the phosphorylation of MAPKs ,IκB and the degradation of IRF3 were detected by the Western Blot assayResults1. Establishment of RAW264.7 cells model of internalization dysfuction(1) MDC ,dynasore and CQ prevented LPS-induced internalization of LPS and TLR4 complex and thus allowed the establishment of RAW264.7 cells model of internalization dysfunction;(2) Application of siRNA technology interfere with clathrin function failed to obtain stable transfected RAW264.7 cell line2. Examination of the activation condition of RAW264.7 cells model of internalization dysfuction(1) RT-PCR and ELISA data indicated that pretreatment of cells with100μM MDC or 80μM dynasore dramatically inhibited the expression of IL-6 (P<0.01);(2) Pretreatment of cells with 20μg/ml CQ significantly inhibited the expression of IL-6 and TNF-α(P<0.05);(3) Results of cytokines arrays showed expression of the major proinflammatory cytokines/chemokines except IL-12p40p70 induced by LPS were significantly affected by pretreatment of MDC and dynasore, while CQ could inhibited the expression of all the cytokines or chemokines ;3. The mechanism study of internalization dysfunction on the activation of RAW264.7 cells(1) Semiquantitative RT-PCR results indicated that the mRNA expression of two key adaptor molecules MyD88 and TRAM involved in the TLR4 signal pathways. MDC and dynasore could effectively decrease the expression of TRAM mRNA stimulated by LPS, whereas MyD88 level was unaffected. However, CQ could effectively decrease the expression of both MyD88 and TRAM mRNA stimulated by LPS;(2) MDC and dynasore pretreatment significantly inhibited late phase activation of NF-κB activation(12h), CQ significantly inhibited both the early phase and late phase activation of NF-κB ;(3) Western blot analysis showed that the phosphorylation of IκBα, JNK, ERK1/2, p38 and the degradation of IRF3 were significantly inhibited by MDC , dynasore and CQ . Conclusions(1) Early internalization of LPS-TLR4 complex was closely related to MyD88-independent signal transduction pathway. While the endosome acidifying maturation was closely related to both TLR4-mediated MyD88-dependent and MyD88-independent signaling pathway.(2) Inhibition of early internalization of LPS-TLR4 complex downregulate MyD88-independent signaling pathways of TLR4 which contribute to inhibit the late phase acvitation of NF-κB, MAPKs and IRF3 and thus significantly suppress macrophage activation; On the other hand, chlorquine supress both MyD88 dependent and independent signaling pathways due to inhibition with endosome acidifying maturation which cause the LPS-TLR4 complex accumulates in endosome and prevent the recycle of TLR4 back to the cell surface.